Antioxidant, anti-inflammatory, anti-radiation, metal chelating compounds and uses thereof

ABSTRACT

Potent compounds having combined antioxidant, anti-inflammatory, anti-radiation and metal chelating properties are described. Short peptides having these properties, and methods and uses of such short peptides in clinical and cosmetic applications are described.

FIELD OF THE INVENTION

The present invention relates to potent compounds having combinedantioxidant, anti-inflammatory, anti-radiation and metal chelatingproperties. More specifically, the present invention relates to shortpeptides having said properties, and to methods and uses of such shortpeptides in clinical and cosmetic applications.

BACKGROUND OF THE INVENTION

The normal functioning of biological systems requires, inter alia,proper balance between formation and elimination of damaging substances.Oxidative stress, for example, represents an imbalance between thelevels of damaging oxidizing species, such as reactive oxygen species(ROS) and reactive nitrogen species (RNS), in a biological system andthe insufficient ability of that biological system to readily neutralizeor eliminate the oxidizing species. Consequences of this stress includebut are not limited to deleterious modification to cellular proteins,lipids and DNA.

Oxidative stress is associated with a wide range of diseases and othermedical conditions, including for example Alzheimer's disease,Parkinson's disease, diabetes and pathologies secondary to diabetes,rheumatoid arthritis, neurodegeneration (particularly in motor neurondiseases), airway inflammation and hyper-responsiveness (for example,asthma), and some skin disorders, such as vitiligo.

In some of these cases, it is unclear whether the oxidative stress isthe cause or the consequence of the medical condition. However, in manycases, lowering the oxidative stress leads to improvement in the diseasemanifestation. In many other cases, lowering the oxidative stress mayprevent the disease outbreak. In addition to pathological conditions,oxidative stress is also known to be involved in some undesiredcomponents of aging.

Thiol (—SH) containing compounds are a type of molecules capable ofneutralizing several types of damaging oxidative species, thus acting asreducing reagents. The activity of this group of compounds is mainly dueto the sulfur atom they comprise which participates in nucleophilicattack on toxic electrophiles, scavenging free radicals, effectingrepair of damaged targets through hydrogen atom donation, altering theredox status of the cell, or affecting gene transcription or proteinfunction.

Thiol containing compounds include natural molecules, produced by allliving organisms including animals and plants, as well as syntheticmolecules. Examples of natural thiol containing antioxidants includeglutathione, which is one of the most potent and important antioxidantsin mammals, thioredoxins and cysteine.

Examples of synthetic thiol containing redox molecules includeN-acetylcysteine amide, as described, for example, in Atlas et al.,(2005) Free Radic Biol Med, Vol. 38(1), pp. 136-45. Another example isN-acetyl-cysteine-proline-cysteine-amide (CB3), described, for example,in Kim et al. (2011) 183(8):1015, which evaluated its protectiveproperties in allergic airway diseases using an ovalbumin(OVA)-inhalation model in mice.

U.S. Pat. No. 5,874,468 discloses brain targeted low molecular weight,hydrophobic antioxidants and use of antioxidants in treatment of centralnervous system neurodegenerative disorders such as Parkinson's,Alzheimer's and Creutzfeldt-Jakob's diseases and in treatment ofconditions of peripheral tissues, such as acute respiratory distresssyndrome, amyotrophic lateral sclerosis, atherosclerotic cardiovasculardisease and multiple organ dysfunction, in which oxidants areoverproduced.

U.S. Pat. No. 6,369,106 discloses a method of reducing oxidative stressin the brain of an organism having a blood brain barrier and sufferingan ischemic brain injury, the method comprising the step ofadministering a compound to the organism, the compound having (a) acombination of molecular weight and membrane miscibility properties forpermitting the compound to cross the blood brain barrier of theorganism; (b) a readily oxidizable chemical group for exertingantioxidation properties; and (c) a chemical make-up for permitting thecompound or its intracellular derivative to accumulate within thecytoplasm of cells.

International Patent Application Publication No. WO 2002/034202discloses an antioxidant compound characterized by (a) a peptideincluding at least three amino acid residues of which at least two arecysteine residues, each having a readily oxidizable sulfhydryl group foreffecting antioxidation; and at least two peptide bonds, each beingcleavable by at least one intracellular peptidase; and (b) a firsthydrophobic or non-charged moiety being attached to an amino terminal ofthe peptide via a first bond and a second hydrophobic or non-chargedmoiety being attached to a carboxy terminal of the peptide via a secondbond, the first hydrophobic or non-charged moiety and the secondhydrophobic or non-charged moiety are selected so as to provide theantioxidant compound with membrane miscibility properties for permittingthe antioxidant compound to cross cellular membranes; wherein cleavageof the at least two peptide bonds by the at least one intracellularpeptidase results in generation of a plurality of antioxidant species,each including one of the cysteine residues having the readilyoxidizable sulfhydryl group and which is also active in effectingantioxidation, thereby providing for a plurality of differentantioxidant species acting in synergy in exerting antioxidation.

Metal chelation is another important aspect in protecting biologicalsystems from harmful substances. Metals, particularly heavy metals, areknown to exert toxic effects when present in inappropriate amounts.Metal ions may generate free radicals reactions, thereby contributing tooxidative stress.

Ionizing radiation such as ultraviolet (UV) light, X rays, gamma rays,neutron beam and proton beam among others, may induce oxidative stress,inflammation, bone marrow damage (resulting in leukopenia,thrombocytopenia and anemia), digestive system damage (including loss ofmicrovilli in the intestine walls) and abundant mutagenic and cytotoxicDNA lesions, which are responsible for the development of benign tumors,cancer (including hematological malignancies) and many other harmfulconditions and disorders. Ionizing radiation promotes the production offree radicals, therefore antioxidants may protect against ionizingradiation-induced damage. It has been shown that antioxidant thiols(N-acetylcysteine amide, glutathione and thioproline) are capable ofprotecting against radiation-induced damage to cellular DNA in humanblood lymphocytes (Tiwari et al. Mutat Res. 2009, 31; 676(1-2):62-8).The various sources of natural ionizing radiation include cosmicradiation, solar radiation (including UV and protons) and high radon gasenvironments. The various sources of artificial ionizing radiationinclude external and internal radiation during medical procedures suchas diagnostic imaging and scanning, injected or swallowed radioactiveisotopes, nuclear medicine and radiation therapy, among others. There isan unmet need for improved compositions directed to the prevention andtreatment of damages caused by ionizing radiation, suitable for oral,topical and systemic administration.

Additionally, there still remains a need for more effective means tohandle conditions associated with excess amounts or excessiveconcentrations of damaging substances in cells. For example, it would behighly beneficial to have antioxidant, anti-inflammatory, anti-allergyand metal chelating compounds, with a combination of high potency, goodstability, bioavailability and sufficient half life to achieve thedesired effects.

SUMMARY OF THE INVENTION

The present invention provides short peptides having antioxidant,anti-inflammatory, anti-allergy, anti-radiation and metal chelatingproperties. The present invention further provides compositions, methodsand uses of such short peptides in clinical and cosmetic applications.

The present invention discloses highly potent peptide compoundscomprising unique dipeptide sequences located between two cysteine aminoacid residues, and further comprising N- and C-terminal modifications.Typically, the amino and carboxy termini are blocked by appropriateblocking groups. Suitable amino terminal blocking groups include, butare not limited to, alkyl and acyl. Suitable carboxy terminal blockinggroups include, but are not limited to, amide, ester and alcohol.Blocking groups exemplified herein include N-acetyl and C-terminal amidegroups.

The peptides of the present invention were found to be highly effectivein reducing inflammatory, as well as allergic responses in vivo, asexemplified hereinbelow. Surprisingly, the peptides of the presentinvention were found to be superior to known thiol-containing molecules,such as N-acetyl-cysteine-proline-cysteine-amide (also known as CB3) andN-acetyl-cysteine-glycine-proline-cysteine-amide (also known as CB4).The peptides of the present invention were shown to affectinflammatory-associated responses in the cellular level, by dramaticallydecreasing nuclear translocation of the nuclear factor-κB (NF-κB), andincreasing cytoplasmic IkappaBalpha concentration in lung cells uponsystemic inflammatory stimulation.

In addition, the peptides of the present invention were found to behighly effective in protecting keratinocytes against hydrogenperoxide-induced cytotoxicity, reducing ROS formation upon LPSadministration in vivo, reversing oxidative stress-inducedphosphorylation of p38 and JNK in model cells, and protecting cellsagainst UVB-induced irradiation, as further exemplified hereinbelow.

The peptides of the present invention are able to cross membranes andenter cells very efficiently. Without wishing to be bound by anyparticular theory or mechanism of action, it is contemplated that the N-and C-termini modifications of the peptides of the present inventionreduce their polarity, thus facilitating the ability of these peptidesto cross cell membranes, enter easily into cells and to accumulatewithin the cells. In addition, the modifications may stabilize andprotect the peptides from degradation, including for example enzymatic,chemical or biochemical breakdown of the molecules.

The properties of the short peptides of the present invention may beutilized in various applications. For example, such peptides may havetherapeutic applications in the treatment and/or prevention of diseasesand disorders associated with oxidative stress, and/or in the treatmentand/or prevention of diseases and disorders associated withinflammation, and/or accumulation of metal ions. In addition, peptidesof the present invention may be used not only as anti-inflammatory butalso as anti-allergic agents. The properties of the peptides of thepresent invention may also be utilized in anti-aging treatments andcosmetic applications, such as cosmetic compositions.

According to one aspect, the present invention provides a peptide havingan amino acid sequence selected from the group consisting of:

(SEQ ID NO: 1) Cys-Lys-Met-Cys, (SEQ ID NO: 2) Cys-Met-Lys-Cys; and(SEQ ID NO: 3) Cys-β-Ala-His-Cys.

In some embodiments, a tetra-peptide is provided, selected from thegroup consisting of:

(SEQ ID NO: 1) Cys-Lys-Met-Cys, (SEQ ID NO: 2) Cys-Met-Lys-Cys; and(SEQ ID NO: 3) Cys-β-Ala-His-Cys.

In some embodiments, the peptide further comprises at least onemodification of the peptide's terminus. According to some embodiments,the peptide comprises an amino-terminal modification. According to otherembodiments, the peptide comprises a carboxy-terminal modification.According to yet other embodiments, the peptide comprises bothamino-terminal and carboxy-terminal modifications. Each possibilityrepresents a separate embodiment of the invention.

In principle, any group suitable for amino terminus modification, andany group suitable for carboxy terminus modification may be used for thepeptide of the present invention.

In some embodiments, the amino terminal modification is an aminoterminal blocking group.

In some typical embodiments, the amino-terminal blocking group isselected from the group consisting of alkyl and acyl. Each possibilityrepresents a separate embodiment of the invention.

In some exemplary embodiments, the amino-terminal blocking group is anacetyl group.

In some embodiments of the present invention, the amino terminalmodification is a moiety that improves the ability of the peptide topenetrate lipid layers and/or improves the ability of the peptide topenetrate into the skin. Such moiety may provide high efficacy topicaladministration. In some exemplary embodiments, the moiety that improvesthat ability of the peptide to penetrate lipid layers and/or improvesits ability to penetrate the skin is a fatty acid. In some embodiments,the fatty acid is selected from the group consisting of palmitic acid,phosphatidic acid, stearic acid, arachidonic acid, docosahexaenoic acid,eicosapentaenoic acid, and oleic acid. Each possibility represents aseparate embodiment of the invention.

In some embodiments, the amino terminal modification is selected fromthe group consisting of an amino terminal blocking group and a fattyacid. Each possibility represents a separate embodiment of theinvention.

In some embodiments, the amino terminal modification is selected fromthe group consisting of alkyl, acyl and a fatty acid.

In some embodiments, the carboxy terminal modification is a carboxyterminal blocking group. In some typical embodiments, the carboxyterminal blocking group is selected from the group consisting of amide,ester and alcohol group. Each possibility represents a separateembodiment of the invention. In some exemplary embodiments, the carboxyterminal blocking group is an amide group.

In some embodiments, the peptide is cleavable by intracellularpeptidases. The cleavage of the peptide upon entry into cells may resultin molecules that exhibit antioxidant, anti-inflammatory and metalchelation properties and activities.

In some additional or alternative embodiments, the N- and/or C-terminalmodifications are hydrolysable by intracellular enzymes. Thus, thesemodifications may be hydrolyzed upon entry of the peptide into cells.

In some embodiments of the present invention, the middle dipeptide,located between the two Cys residues, is selected from the groupconsisting of Lys-Met and Met-Lys. Each possibility represents aseparate embodiment of the invention. Lysine and methionine areprecursors for the biosynthesis of carnitine molecules. In living cells,carnitine is required for the transport of fatty acids from the cytosolinto the mitochondria where the breakdown of lipids takes place duringthe generation of metabolic energy. Carnitine is also known to havestrong antioxidant activity. As noted above, the peptide may be cleavedby peptidases upon entry into cells. In some embodiments, the peptideundergoes cleavage that results in the release of lysine and methionine.In some embodiments, administration of the peptide may promote theformation of carnitine and may increase the amount and concentration ofcarnitine in cells. Such peptide may be used in a method for increasingthe amount and concentration of carnitine in cells.

In some embodiments of the present invention, the dipeptide β-Ala-His islocated between the two Cys residues. The dipeptide β-Ala-His(β-alanyl-L-histidine) is known as carnosine. Carnosine is capable ofperforming a variety of functions, including anti-oxidation,anti-glycation, pH buffering and chelation of divalent metal cations. Insome embodiments, the peptide undergoes intracellular cleavage thatresults in the release of free carnosine. In some embodiments,administration of the peptide may increase the amount and concentrationof carnosine in cells. Such peptide may be used in a method forincreasing the amount and concentration of carnosine in cells.

In some exemplary embodiments, the peptideN-acetyl-Cys-Lys-Met-Cys-amide (SEQ ID NO: 4) is provided.

In additional exemplary embodiments, the peptideN-acetyl-Cys-Met-Lys-Cys-amide (SEQ ID NO: 5) is provided.

In yet additional exemplary embodiments, the peptideN-acetyl-Cys-β-Ala-His-Cys-amide (SEQ ID NO: 6) is provided.

In some embodiments of the present invention, the peptide has anactivity selected from the group consisting of antioxidant,anti-inflammatory, anti-allergy, anti-radiation, metal chelation orcombinations thereof. Each possibility represents a separate embodimentof the invention.

In some embodiments of the present invention, the peptide has ananti-inflammatory activity. In some embodiments of the presentinvention, the peptide has an anti-allergic activity. In someembodiments of the present invention, the peptide has an anti-oxidantactivity.

In some embodiments of the present invention, the peptide is in the formof a salt. In some embodiments, the salt is selected from the groupconsisting of trifluoroacetic acid (TFA), acetate and citrate salts.Each possibility represents a separate embodiment of the invention.

According to another aspect, the present invention provides acomposition comprising a peptide of the present invention or a saltthereof.

In some embodiments of the present invention, the peptide is formulatedas a pharmaceutical composition. According to these embodiments, thecomposition further comprises a pharmaceutically acceptable diluent,solvent, excipient or carrier.

In some embodiments of the present invention, the peptide is formulatedas a cosmetic composition. According to these embodiments, thecomposition further comprises a cosmetically acceptable diluent,solvent, excipient or carrier.

Any suitable route of administration may be used for the composition ofthe present invention, including but not limited to local and systemicroutes. Systemic administration includes all enteral and all parenteralroutes. Non-limiting examples of suitable administration routes includetopical application, oral, rectal, transmucosal such as transnasal andbuccal, intravenous, intramuscular, transdermal, subcutaneous,intradermal, intravitreal, intravesicular and inhalation routes.

Thus, in some embodiments, the composition of the present invention isformulated for topical administration. In other embodiments, thecomposition is formulated for systemic administration.

The compositions of the present invention are useful for the treatmentor prevention of diseases and disorders associated with oxidativestress. The compositions of the present invention are also useful forthe treatment or prevention of diseases and disorders associated withinflammation and/or allergy. In addition, the compositions of thepresent invention are useful for the treatment or prevention of diseasesand disorders associated with the presence or accumulation of advancedglycation end products, also known as non-enzymatic glycosylationproducts. In addition, the compositions of the present invention areuseful for the treatment or prevention of diseases and disordersassociated with the presence or accumulation of metal ions. Thecompositions of the present invention are also useful for the treatmentor prevention of damages caused by ionizing radiation.

Thus, according to another aspect, the present invention provides amethod for treating a disease or disorder associated with at least oneof oxidative stress, allergy or inflammation, the method comprisingadministering to a subject in need a composition comprising a peptide ofthe present invention or a salt thereof. According to certainembodiments, the method comprises administering mixtures of peptides ofthe invention.

In some embodiments of the present invention, a pharmaceuticalcomposition is provided, comprising at least one peptide of the presentinvention or at least one salt thereof as an active ingredient, for usein the treatment of a disease or disorder associated with at least oneof oxidative stress, allergy (chronic and/or acute flare) orinflammation (chronic and/or acute flare).

In some embodiments, the disease or disorder is associated withoxidative stress. In some embodiments, the disease or disorderassociated with oxidative stress is selected from the group consistingof Alzheimer's disease, Parkinson's disease, diabetes, rheumatoidarthritis, ischemia-reperfusion injury and vitiligo. Each possibilityrepresents a separate embodiment of the invention. The diabetes mayinclude type I and type II diabetes. Each possibility represents aseparate embodiment of the invention.

In some embodiments, the disease or disorder is associated withinflammation. In some embodiments, the disease or disorder associatedwith inflammation is an auto-immune disease. In some embodiments, thedisease or disorder associated with inflammation is selected from thegroup consisting of acute inflammation, rheumatoid arthritis, aninflammatory bowel disease and atherosclerosis. Each possibilityrepresents a separate embodiment of the invention.

In some embodiments, the disease or disorder is associated with allergy.In some embodiments, the disease or disorder associated with allergy isselected from the group consisting of an allergic airway disease,allergic rhinitis, eczema, dermatitis, a gastrointestinal food allergyand an ocular allergy. Each possibility represents a separate embodimentof the invention. In some embodiments, the allergic disease is asthma.

According to another aspect, the present invention provides a method fortreating a disease or disorder associated with the presence oraccumulation of advanced glycation end products, the method comprisingadministering to a subject in need thereof a composition comprising asactive ingredient a peptide of the present invention or a salt thereof.According to certain embodiments, the method comprises administeringmixtures of peptides of the invention.

In some embodiments of the present invention, a pharmaceuticalcomposition is provided, comprising at least one peptide of the presentinvention or at least one salt thereof as an active ingredient, for usein the treatment and/or prevention of diseases or disorders associatedwith the presence or accumulation of advanced glycation end products.

In some embodiments, the disease or disorder associated with thepresence or accumulation of advanced glycation end products is selectedfrom the group consisting of diabetes, cataract, heart failure,hypertension and callused skin. Each possibility represents a separateembodiment of the invention. The diabetes may include type I and type IIdiabetes. Each possibility represents a separate embodiment of theinvention.

In some embodiments of the present invention, the disease or disorder isa skin condition and/or cosmetic issue. In some exemplary embodiments,the skin condition is vitiligo.

In some embodiments, the present invention provides the use of a peptideof the present invention or a salt thereof as an anti-inflammatoryagent.

In additional embodiments, the present invention provides the use of apeptide of the present invention or a salt thereof as an anti-allergicagent.

In additional embodiments, the present invention provides the use of apeptide of the present invention or a salt thereof as an antioxidant.

According to another aspect, the present invention provides a method fortreating a disease or disorder associated with the presence oraccumulation of metal ions, the method comprising administering to asubject in need thereof a pharmaceutical composition comprising as anactive ingredient a peptide of the present invention or a salt thereof.

In some embodiments of the present invention, a pharmaceuticalcomposition is provided, comprising at least one peptide of the presentinvention or at least one salt thereof as an active ingredient, for usein the treatment of a disease or disorder associated with the presenceor accumulation of metal ions.

In some embodiments, the disease or disorder associated with thepresence or accumulation of metals ions is selected from the groupconsisting of a liver disorder, Alzheimer's disease, heart attack(myocardial infarction), stroke and a prion disease. Each possibilityrepresents a separate embodiment of the invention.

The antioxidant properties of the peptides of the present invention maybe utilized in cosmetic applications, for skin care and protection. Forexample, the peptides of the present invention may be utilized asanti-aging agents.

Thus, according to yet another aspect, the present invention provides amethod for slowing the aging process of the human skin, reducing thesigns of aging of the human skin or both, the method comprising applyingto the skin a cosmetic composition comprising the peptide of the presentinvention or a salt thereof.

In some embodiments, slowing the aging process of the human skin andreducing the signs of aging of the human skin encompass improvement ofthe skin tone, reduction of wrinkles, removal of lines, promotion ofskin firmness and/or reduction of skin sensitivity and irritability.

In some embodiments, a cosmetic composition is provided, comprising apeptide of the present invention or a salt thereof as active ingredient,for use in slowing the aging process of the human skin, reducing thesigns of aging of the human skin or both.

According to yet another aspect, the present invention provides a methodfor the prevention or treatment of a disorder or harmful effects causedby ionizing radiation comprising administering to a subject in needthereof a composition comprising a peptide of the present invention or asalt thereof. According to some embodiments, the method comprisesadministering mixtures of peptides of the present invention. Accordingto some embodiments the compositions are administered in order toprevent or reduce at-least some of the harmful effects of radiation.According to other embodiments the compositions are administered toameliorate at-least some of the harmful effects of prior exposure toradiation.

According to yet another aspect the present invention provides acomposition for the prevention or treatment of at-least some of theharmful effects caused by ionizing radiation comprising a peptide of thepresent invention or a salt thereof. According to some embodiments, thecomposition comprises a mixture of peptides of the present invention.

According to some embodiments, ionizing radiation is selected from thegroup consisting of x-rays radiation, gamma-radiation, ultravioletradiation, thermal radiation, nuclear radiation, cosmic radiation, or acombination thereof. Each possibility represents a separate embodimentof the invention. In some embodiments, the ionizing radiation isultraviolet radiation. In some exemplary embodiments, the ultravioletradiation is UVB.

According to some embodiments, the composition is administered within 24hours before the exposure to the ionizing radiation or at about the timeof exposure to the ionizing radiation and/or within 24 hours after theexposure to the ionizing radiation. According to alternativeembodiments, the composition is administered within 12 hours, within sixhours or within three hours or less before and/or after the exposure tothe ionizing radiation. Each possibility represents a separateembodiment of the present invention.

These and further aspects and features of the present invention willbecome apparent from the figures, detailed description, examples andclaims which follow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Effect of peptides on carrageenan-induced hind paw swelling.

FIG. 2. Effect of peptides on malondialdehyde (MDA) activity.

FIG. 3A-F. Effect of the peptides on nuclear translocation of NF-κB andcytoplasmic stability of I-kappaBalpha. The gels show representation of4 samples. The densitometry results reflect all samples (up to 8 samplesfor each peptide).

FIG. 4A-C. Effect of peptides on ovalbumin-induced hypersensitivity inmice—analysis of blood and peritoneal exudates samples.

FIG. 5A-H. Effect of peptides on ovalbumin-induced hypersensitivity inmice—analysis of peritoneal exudate samples.

FIG. 6A-D. Effect of peptides on hydrogen peroxide-induced HaCaTkeratinocyte cytotoxicity. 6A, C—effect of the tested peptide on HaCaTcell viability (no hydrogen peroxide). 6B, D—effect of the testedpeptide on hydrogen peroxide-induced cytotoxicity in HaCaT cells.

FIG. 7A-C. Effect of the peptides on UVB-induced cell cytotoxicity inHaCaT cells.

FIG. 8. Effect of DY-66 on liver inflammation in a mouse model of liverinflammation induced by injection (i.v.) of Concanavalin A (Con-A).

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses short peptides having antioxidant,anti-inflammatory, anti-allergy, anti-radiation and metal chelatingproperties, and their use in therapeutic, preventive and cosmeticapplications.

The peptides of the present invention show a desirable combination ofproperties such as ability to enter cells, stability inside the cells,high potency and low toxicity.

Definitions

As used herein “peptide” indicates a sequence of amino acids linked bypeptide bonds. The term “tetra-peptide” indicates a peptide composed offour amino acids. The peptides of the present invention are typicallyutilized in a linear form, although it will be appreciated that in caseswhere cyclization does not severely interfere with peptidecharacteristics, cyclic forms of the peptide can also be utilized.

The term “amino acid” refers to compounds, which have an amino group anda carboxylic acid group, preferably in a 1,2- 1,3-, or 1,4-substitutionpattern on a carbon backbone. The term encompasses natural, non-naturaland/or chemically modified amino acid residues. Natural amino acidsinclude those found in proteins, which are L-amino acids. Non-naturaland/or chemically modified amino acids include, for example, thecorresponding N-methyl amino acids, side chain modified amino acids andthe biosynthetically available amino acids which are not found inproteins (e.g., 5-hydroxy-lysine). The amino acid residues arerepresented throughout the specification and claims by either one orthree-letter codes, as is commonly known in the art. The amino acidsused in this invention are those which are available commercially or areavailable by routine synthetic methods. Certain residues may requirespecial methods for incorporation into the peptide, and eithersequential, divergent or convergent synthetic approaches to the peptidesequence are useful in this invention.

Also included within the scope of the invention are salts of thepeptides, and derivatives of the peptides of the invention.

As used herein the term “salts” refers to salts of carboxyl groups andto acid addition salts of amino groups of the peptide molecule. Salts ofcarboxyl groups may be formed by means known in the art and includeinorganic salts, for example sodium, calcium, ammonium, ferric or zincsalts, and the like, and salts with organic bases such as salts formedfor example with amines such as triethanolamine, piperidine, procaine,and the like. Acid addition salts include, for example, salts withmineral acids such as, acetic acid or oxalic acid. Additional examplesof suitable salts include trifluoroacetic acid (TFA), acetate andcitrate salts.

Esters and amides of carboxy groups and acyl and alkyl derivatives ofamino groups may be introduced into the molecule by reacting targetedamino acid residues of the peptide with an organic derivatizing agentthat is capable of reacting with terminal residues. Preferred chemicalderivatives include peptides that have been C-termini amidated orN-termini acetylated.

“Derivatives” of the peptides of the invention as used herein coversderivatives which may be prepared from the functional groups which occuras side chains on the residues or the N- or C-terminal groups, by meansknown in the art, and are included in the invention as long as theyremain pharmaceutically/cosmetically acceptable, i.e., they do notdestroy the activity of the peptide, do not confer toxic properties oncompositions containing it and do not adversely affect the antigenicproperties thereof. These derivatives may, for example, includealiphatic esters of the carboxyl groups, amides of the carboxyl groupsproduced by reaction with ammonia or with primary or secondary amines,N-acyl derivatives of free amino groups of the amino acid residuesformed by reaction with acyl moieties (e.g., alkanoyl or carbocyclicaroyl groups).

“Permeability” refers to the ability of an agent or substance topenetrate, pervade, or diffuse through a barrier, membrane, or a skinlayer. A “cell permeability”, “cell-penetration” or“permeability-enhancing” moiety refers to any molecule known in the artwhich is able to facilitate or enhance penetration of molecules throughmembranes. Non-limitative examples include: hydrophobic moieties such aslipids, fatty acids, steroids and bulky aromatic or aliphatic compounds.The permeability-enhancing moiety may be connected to any position inthe peptide moiety, directly or through a spacer, preferably to theamino or carboxy terminus of the peptide moiety.

As used herein, the term “diseases and disorders associated withoxidative stress” refers to diseases and disorders caused and/orexacerbated by oxidative stress, or diseases and disorders characterizedby abnormal increased oxidative stress.

As used herein, the terms “diseases and disorders associated withallergy”, “allergic diseases” and “allergic disorders” are usedinterchangeably and refer to diseases and disorders characterized byhypersensitivity and hyper-responsiveness of the immune system to one ormore foreign substances that are normally harmless (termed allergens).Allergic reactions are typically characterized by excessive activationof mast cells, basophils and eosinophils by Immunoglobulin E (IgE)antibodies.

As used herein, the terms “diseases and disorders associated withinflammation” and “inflammatory diseases/disorders” are usedinterchangeably and refer to diseases or disorder characterized byinflammation. The term encompasses both acute and chronic inflammation.

As used herein, the term diseases and disorders associated with thepresence or accumulation of advanced glycation end-products (AGE) refersto diseases and disorders caused and/or exacerbated by AGE, or diseasesand disorders characterized by accelerated formation of AGE and/orabnormal increased levels of AGE.

The term “advanced glycation end-products” (AGE) refers to aheterogeneous group of molecules formed from non-enzymatic reactions ofreducing sugars (typically glucose or fructose) with free amino groupsof proteins, lipids and nucleic acids. Certain reactive or precursorAGEs are able to form covalent crosslinks between proteins, which alterstheir structure and function,

As used herein, the term “diseases and disorders associated withpresence or accumulation of metal ions” refers to diseases and disorderscaused and/or exacerbated by the presence or accumulation of metal ions,or diseases and disorders characterized by abnormal increased amounts orconcentrations of metal ions.

As used herein, “treating” and “treatment”, refers to reduction,amelioration or even elimination of at least some of the symptomsassociated with the relevant disease. The term may include reduction ofoxidative stress; inhibition or reduction of inflammation; inhibition orreduction of allergic reactions; reduction in the level or concentrationof advanced glycation end products and/or neutralizing or loweringadvanced glycation end products damage; and/or reduction in the level ofdamaging metals and/or neutralizing or lowering metal ion damage.

Peptides of the Present Invention

According to one aspect, the present invention provides a peptide havingan amino acid sequence selected from the group consisting of:

(SEQ ID NO: 1) Cys-Lys-Met-Cys, (SEQ ID NO: 2) Cys-Met-Lys-Cys,(SEQ ID NO: 3) Cys-β-Ala-His-Cys, ((SEQ ID NO: 7) Cys-γ-Glu-Cys-Cys(SEQ ID NO: 8) Cys-γ-Glu-Cys.

Each possibility represents a separate embodiment of the invention.

In some embodiments, the peptide is selected from the group consistingof:

(SEQ ID NO: 1) Cys-Lys-Met-Cys, (SEQ ID NO: 2) Cys-Met-Lys-Cys; and(SEQ ID NO: 3) Cys-β-Ala-His-Cys.

It is understood that Cys represents the amino-acid cysteine; Lysrepresents the amino-acid lysine; Met represents the amino-acidmethionine; β-Ala represents the amino-acid β-alanine; and γ-Glurepresents the amino-acid γ-glutamic acid/γ-glutamate.

In some embodiments, the peptide further comprising at least onemodification selected from the group consisting of an amino-terminalmodification and a carboxy-terminal modification. According to theseembodiments, the peptide is selected from the group consisting of:

(SEQ ID NO: 9) Z- Cys-Lys-Met-Cys- Y, (SEQ ID NO: 10)Z- Cys-Met-Lys-Cys- Y; and (SEQ ID NO: 11) Z- Cys-β-Ala-His-Cys- Y,

wherein Z represents an amino terminal modification and Y represents acarboxy terminal modification.

In some embodiments, the N- and C-termini modifications reduce thepolarity of the peptides of the present invention, thus facilitating theability of these peptides to cross cell membranes, enter easily intocells and accumulate within the cells. In addition, modifications of thepeptide termini may improve bio-stability, for example by blocking theaction of peptidases.

The amino and carboxy termini modifications may be chosen from any aminoand carboxy termini modifications conventionally used in the art ofpeptide chemistry, which will not adversely affect the activities of thepeptide.

In some embodiments, the amino terminal modification comprises additionof an amino terminal blocking group.

Blocking of the N terminus may be performed, for example, by alkylationor acylation, using methods well known in the art. Non-limiting examplesof suitable N-terminal blocking groups include C₁-C₅ branched orunbranched alkyl groups, acyl groups such as formyl and acetyl groups,and substituted forms thereof, such as the acetamidomethyl (Acm) group.Each possibility represents a separate embodiment of the invention.

In some embodiments of the present invention, the amino terminalmodification comprises covalently linking to the N-terminus of thepeptide a moiety that improves the ability of the peptide to penetratelipid layers and/or improves the ability of the peptide to penetrateinto the skin. Such moiety may provide high efficacy topicaladministration. In some exemplary embodiments, the moiety is a fattyacid. The fatty acid may be selected from the group consisting ofpalmitic acid, phosphatidic acid, stearic acid, arachidonic acid,docosahexaenoic acid, eicosapentaenoic acid and oleic acid. Eachpossibility represents a separate embodiment of the invention.

In some typical embodiments, the amino terminal modification is selectedfrom the group consisting of acetyl, alkyl, acyl and a fatty acid. Eachpossibility represents a separate embodiment of the invention.

In some embodiments, the carboxy terminal modification is a carboxyterminal blocking group.

Blocking of the C terminus may be performed, for example, by amidation,reduction or esterification, using methods well known in the art.Non-limiting examples of suitable C-terminal blocking groups includeamide, ester, and alcohol groups. Each possibility represents a separateembodiment of the invention.

Upon entry of the peptides into cells they may undergo cleavage byintracellular peptidases. The cleavage may result in molecules thatstill exert antioxidant, anti-inflammatory, anti-glycation and metalchelation properties and activities.

In addition, the N- and/or C-termini modifications of the peptides maybe hydrolyzed, which may result in their accumulation in the cytosol.

In some embodiments the middle dipeptide, located between the two Cysresidues, is selected from the group consisting of Lys-Met and Met-Lys.In some embodiments, a peptide is provided, selected from the groupconsisting of Cys-Lys-Met-Cys (SEQ ID NO: 1) and Cys-Met-Lys-Cys (SEQ IDNO: 2).

In some exemplary embodiments, the peptideN-acetyl-Cys-Lys-Met-Cys-amide (SEQ ID NO: 6) is provided.

In additional exemplary embodiments, the peptideN-acetyl-Cys-Met-Lys-Cys-amide (SEQ ID NO: 7) is provided.

In some embodiments the dipeptide β-Ala-His, is located between the twoCys residues. In some embodiments, a peptide Cys-β-Ala-His-Cys (SEQ IDNO: 3) is provided. In some exemplary embodiments, the peptideN-acetyl-Cys-β-Ala-His-Cys-amide (SEQ ID NO: 8) is provided.

In some embodiments, the dipeptide γ-Glu-Cys is located between the twoCys residues. In some embodiments, γ-Glu is located between the two Cysresidues.

In some exemplary embodiments, a peptide is provided, selected from thegroup consisting of N-acetyl-Cys-γ-Glu-Cys-Cys-amide (SEQ ID NO: 12) andN-acetyl-Cys-γ-Glu-Cys-amide (SEQ ID NO: 13).

In some embodiments, the peptide itself and/or its metabolites have anactivity selected from the group consisting of antioxidant,anti-inflammatory, anti-allergy and metal chelating or a combinationthereof. Each possibility represents a separate embodiment of theinvention.

The peptides of the present invention may be capable of chelating a widevariety of metal ions. Non-limiting examples of metal ions include Cu,Fe, Cd, Zn, Mg, Hg, Pb, As, Tl, Au. In some embodiments, the peptide iscapable of chelating at least one metal ion selected from the groupconsisting of Cu and Fe. Each possibility represents a separateembodiment of the invention.

In some embodiments, the peptide has an anti-inflammatory activity. Inadditional embodiments, the peptide has an anti-allergic activity.

The peptides of the present invention may be synthesized by anytechnique known to those skilled in the art of peptide synthesis. Thesemethods include solid phase as well as solution phase synthesis methods.

Solid phase peptide synthesis procedures are well known in the art andfurther described by John Morrow Stewart and Janis Dillaha Young, SolidPhase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

A skilled artesian may synthesize any of the peptides of the presentinvention by using an automated peptide synthesizer using standardchemistry such as, for example, t-Boc or Fmoc chemistry.

The methods include exclusive solid phase synthesis, partial solid phasesynthesis methods, fragment condensation, classical solution synthesis.

Synthetic peptides can be purified by preparative high performanceliquid chromatography (Creighton T. (1983) Proteins, structures andmolecular principles. WH Freeman and Co. N.Y.) and the composition ofwhich can be confirmed via amino acid sequencing. Some of the peptidesof the invention, which include only natural amino acids, may further beprepared using recombinant DNA techniques known in the art. Theconjugation of the peptidic and permeability moieties may be performedusing any methods known in the art, either by solid phase or solutionphase chemistry. Some of the compounds of the present invention mayconveniently be prepared using solution phase synthesis methods. Othermethods known in the art to prepare compounds like those of the presentinvention can be used and are comprised in the scope of the presentinvention.

The permeability-enhancing moiety of the present invention may beconnected to any position in the peptide moiety, directly or through aspacer. According to a specific embodiment, the cell-permeability moietyis connected to the amino terminus of the peptide moiety. The optionalconnective spacer may be of varied lengths and conformations comprisingany suitable chemistry including but not limited to amine, amide,carbamate, thioether, oxyether, sulfonamide bond and the like.Non-limiting examples for such spacers include amino acids, sulfoneamide derivatives, amino thiol derivatives and amino alcoholderivatives.

Cyclic versions of the peptides disclosed herein are also within thescope of the present invention. Cyclization of peptides may take placeby any means known in the art, for example through free amino andcarboxylic groups present in the peptide sequence, or through aminoacids or moieties added for cyclization. Non limiting examples ofcyclization types are: side chain to side chain cyclization, C-to-Nterminal cyclization, side chain to terminal cyclization, and any typeof backbone cyclization incorporating at least one N-ω-substituted aminoacid residue/s as described for example in WO 95/33765.

Other methods known in the art to prepare peptides like those of thepresent invention can be used and are within the scope of the presentinvention.

In some embodiments, the peptide is in the form of a salt. Non-limitingexamples of suitable salts include trifluoroacetic acid (TFA), acetateand citrate salts.

Compositions of the Present Invention

According to another aspect, the present invention provides acomposition comprising the peptide of the present invention or a saltthereof.

In some embodiments, the composition is formulated as a pharmaceuticalcomposition. According to these embodiments, the composition furthercomprises a pharmaceutically acceptable diluent, excipient or carrier.

In other embodiments, the composition is formulated as a cosmeticcomposition. According to these embodiments, the composition furthercomprises a cosmetically acceptable diluent, excipient or carrier.

In some embodiments, the composition further comprises at least one moreactive ingredient.

In some embodiments, a pharmaceutical composition is provided,consisting of the peptide of the present invention as an activeingredient. In additional embodiments, a cosmetic composition isprovided, consisting of the peptide of the present invention as anactive ingredient.

The peptide of the present invention or a salt thereof, and optionallyadditional one or more active ingredients, are present in thecompositions of the present invention in an amount effective to achievethe intended purpose, for example, in an amount effective to treat acertain disease.

Any suitable route of administration may be used for the composition ofthe present invention, including but not limited to local and systemicroutes. Systemic administration includes all enteral and all parenteralroutes. Non-limiting examples of suitable administration routes includetopical application, oral, rectal, transmucosal such as transnasal andbuccal, intravenous, intramuscular, transdermal, subcutaneous,intradermal, intravitreal, intravesicular and inhalation routes.

Thus, in some embodiments, the composition of the present invention isformulated for topical administration. In other embodiments, thecomposition is formulated for systemic administration.

Cosmetic and pharmaceutical compositions of the present invention may beformulated in conventional manners. The proper formulation is dependentupon the route of administration chosen.

In some embodiments, the compositions of the present invention areformulated for topical use. Non-limiting examples of formulations fortopical use include cream, ointment, lotion, gel, foam, suspension,aqueous or cosolvent solutions, salve, liposome and sprayable liquidform. The composition may also form part of a patch for transdermalapplication. Other suitable topical product forms for the compositionsof the present invention include, for example, emulsion, mousse, lipbalm, lip gloss, lotion, mask, pomade, solution and serum.

In some embodiments, the cosmetic or pharmaceutical compositions areformulated in the form of a solid or soft gel, for example, anaqueous-alcoholic gel and a clear gel. Typically, the aqueous phasecomprises one or more gelling agents, for example cellulose gellingagents, or synthetic gelling agents.

In some embodiments, the compositions of the present invention areformulated for oral administration. Non-limiting examples offormulations for oral administration include tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for oral ingestion by a patient. Suitable carriers for oraladministration are well known in the art. Compositions for oral use canbe made using a solid excipient, optionally grinding the resultingmixture, and processing the mixture of granules, after adding suitableauxiliaries as desired, to obtain tablets or dragee cores. Non-limitingexamples of suitable excipients include fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol, cellulosepreparations such as, maize starch, wheat starch, rice starch, potatostarch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, and sodium carbomethylcellulose, and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodiumalginate, may be added.

For administration by injection, the active ingredients of thecomposition may be formulated in aqueous solutions, for example inphysiologically compatible buffers including but not limited to Hank'ssolution, Ringer's solution, or physiological salt buffer. Formulationsfor injection may be presented in unit dosage forms, for example, inampoules, or in multi-dose containers with, optionally, an addedpreservative. The compositions may be in the form of suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing, and/or dispersingagents. Non-limiting examples of suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters such as ethyl oleate, triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the activeingredients, to allow for the preparation of highly concentratedsolutions. Alternatively, the active ingredient may be in powder formfor constitution with a suitable vehicle, for example, a sterile,pyrogen-free, water-based solution, before use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation route, the active ingredients areconveniently delivered in the form of an aerosol spray presentation froma pressurized pack or a nebulizer with the use of a suitable propellant,for example, dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane, or carbon dioxide. In the case of apressurized aerosol, the dosage may be determined by providing a valveto deliver a metered amount. Capsules and cartridges of, for example,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base, such as lactose or starch.

In some embodiments, the compositions of the present invention areformulated for rectal administration, for example, as suppositories orretention enemas, using, for example, conventional suppository basessuch as cocoa butter or other glycerides.

The exact formulation, route of administration, and dosage can be chosenby the individual physician in view of the patient's condition.

In some embodiments, the composition further comprises at least oneadditive useful in the cosmetic and pharmaceutical fields, including,but not limited to fats, emulsifiers and co-emulsifiers, hydrophilic orlipophilic gelling agents, colorants, fragrances, emollients,humectants, preservatives, vitamins, chelators, solvents, fillers,thickeners, hydrophilic and lipophilic filters, dyestuffs, neutralizers,penetration-enhancing agents and polymers.

Non-limiting examples of suitable fats include mineral oils, oils ofanimal origin (lanolin), synthetic oils (isopropyl myristate,octyldodecyl, isostearyl isostearate, decyl oleate or isopropylpalmitate), silicone oils (cyclomethicone or dimethicone) andfluorinated oils. Fatty alcohol, fatty acids, waxes and gums, notablysilicone gums and elastomers can also be used as fats.

Non-limiting examples of suitable emulsifiers and co-emulsifiers includepolyglycerol fatty acid esters, sucrose fatty acid esters, sorbitanefatty acid esters, oxyethylene sorbitan fatty acid esters, PEG fattyalcohol ethers, glycerol fatty acid esters, alkyl sulphates, alkyl ethersulphates, alkyl phosphates, alkyl polyglucosides and dimethiconecopolyols.

Non-limiting examples of suitable hydrophilic gelling includecarboxyvinyl polymers (carbomer), acrylic copolymers such asacrylate/alkylacrylate copolymers, polyacrylamids, polysaccharides suchas xanthan gum, guar gum, natural gums such as cellulose gum andderivatives, clays and 2-acrylamido-2-methylpropane acid copolymers.

Non-limiting examples of suitable lipophilic gelling agents includemodified clays such as bentones, fatty acid metal salts, hydrophobicsilica and ethylcellulose.

Non-limiting examples of suitable fillers include talc, kaolin, mica,serecite, magnesium carbonate, aluminum silicate and organic powderssuch as nylon.

Non-limiting examples of suitable dyestuffs include lipophilic dyes,hydrophilic dyes, pigments and mother-of-pearl commonly used in cosmeticor dermatological compositions, and their mixtures.

Non-limiting examples of suitable neutralizers include soda,triethanolamine, aminomethyl propanol and potassium hydroxide.

Non-limiting examples of suitable penetration enhancing agents includealcohols and glycols (ethanol and propylene glycol), ethoxydiglycol,alcohols and fatty acids (oleic acid), fatty acid esters and dimethylisosorbide.

Non-limiting examples of preservatives compatible with cosmetic andpharmaceutical compositions include benzoic acid, its salts and esters,sorbic acid and its salts, parabens and their salts, triclosan,imidazolidinyl urea, phenoxyethanol, DMDM hydantoin, diazolidinyl ureaand chlorphenesin.

Conventionally, the filters are UVA and UVB filters. Non-limitingexamples of suitable UVA and UVB filters include organic filters such asbenzophenone-3, butyl methoxydibenzoyl methane, octocrylene, octylmethoxycinnamate, 4-methylbenzylidene camphor, octyl salicylate,terephthalylidene dicamphor sulfonic acid and drometrizole trisiloxane,and non-organic filters such as titanium oxide and zinc oxide.

Non-limiting examples of suitable solvents include water, ethanol,glycerin, propylene glycol, butylene glycol and sorbitol.

The quantities of these various additives are those conventionally usedin cosmetic and pharmaceutical preparations as is known to a personskilled in the art.

Methods and Uses of the Present Invention

The combination of antioxidant, anti-inflammatory, anti-allergy andmetal chelation activities of the short peptides of the presentinvention render such peptides particularly useful for the treatment ofvarious diseases.

For example, the compositions of the present invention may be useful forthe treatment of diseases associated with oxidative stress, diseasesassociated with the presence or accumulation of advanced glycation endproducts, diseases associated with the presence or accumulation of metalions and diseases associated with inflammation and/or allergy. Eachpossibility represents a separate embodiment of the invention.

In some embodiments, the methods of the present invention compriseadministering a composition comprising at least one tetra-peptide of thepresent invention.

Thus, according to another aspect, the present invention provides amethod for treating and/or preventing a disease or disorder associatedwith at least one of oxidative stress, allergy or inflammation, themethod comprising administering to a subject in need a compositioncomprising a peptide of the present invention or a salt thereof.

According to certain embodiments, the methods comprise administeringmixtures of peptides of the invention.

In some embodiments, the present invention provides the use of a peptideof the present invention or a salt thereof for the manufacture of amedicament for the treatment and/or prevention of a disease or disorderassociated with at least one of oxidative stress, allergy orinflammation.

In some embodiments, a pharmaceutical composition is provided,comprising a peptide of the present invention or a salt thereof asactive ingredient, for use in the treatment and/or prevention of adisease or disorder associated with at least one of oxidative stress,allergy or inflammation.

In some embodiments of the present invention, the relevant diseases ordisorders are skin conditions and cosmetic issues.

In some embodiments, the disease or disorder is associated withoxidative stress. Examples of diseases and disorders associated withoxidative stress that may be treated using the peptides of the presentinvention include, but are not limited to, Alzheimer's disease,Parkinson's disease, diabetes (type I and type II), rheumatoidarthritis, ischemia-reperfusion injury (for example damage to heart andbrain tissues) and vitiligo. Each possibility represents a separateembodiment of the invention.

In some exemplary embodiments, the disease is vitiligo. According tothese embodiments, a method is provided for treating vitiligo in asubject suffering therefrom, the method comprising applying to the skinof the subject a pharmaceutical composition comprising as activeingredient the peptide of the present invention or a salt thereof.

Vitiligo is a chronic disorder that causes depigmentation of patches ofskin due to destruction of melanocytes and/or impairment in melanocytesfunction. Vitiligo may arise from a number of causes, such as genetic,autoimmune, neurotrophic and toxic factors. In addition, oxidativestress was found to be involved. Handling vitiligo usually includecosmetic camouflage, but the main goal is to stop or slow theprogression of pigment loss and, if possible, to return some color tothe skin. Current treatments for vitiligo include, for example,application of corticosteroid creams and immunomodulator creams,ultraviolet (UV) light therapy and sometimes melanocyte transplantationby surgery. Such treatments are usually long, cause physical discomfortand associated with a number of adverse effects. Without wishing to bebound by any particular theory or mechanism, it is contemplated thattreatment of vitiligo by the method of the present invention involvesneutralization of the accumulated hydrogen peroxide by the peptides ofthe present invention. The method of the present invention thus suggestsa safe and efficient treatment for vitiligo.

In some embodiments, the disease or disorder is associated withinflammation. In some embodiments, the disease or disorder associatedwith inflammation is an auto-immune disease. Examples of diseases anddisorders associated with inflammation that may be treated using thepeptides of the present invention include, but are not limited to acuteinflammation, rheumatoid arthritis, an inflammatory bowel disease andatherosclerosis.

In some embodiments, the disease or disorder is associated with allergy.Examples of diseases and disorders associated with allergy that may betreated using the peptides of the present invention include, but are notlimited to, allergic airway disease, allergic rhinitis, eczema,dermatitis, a gastrointestinal food allergy and an ocular allergy.

In some embodiments, the disease is an inflammatory, allergic airwaydisease (chronic and/or acute flare). In some exemplary embodiments, thedisease is asthma. According to these embodiments, a method is providedfor treating asthma in a subject, the method comprising administering tothe subject a pharmaceutical composition comprising as active ingredientthe peptide of the present invention or a salt thereof.

Asthma is a chronic inflammatory disorder of the airways characterizedby an associated increase in airway responsiveness to inhaled allergensand nonspecific stimuli. A significant amount of data shows an increaseof oxidative stress in allergic airway diseases and indicates apotential role of ROS in pathogenesis of the diseases.

According to another aspect, the present invention provides a method fortreating and/or preventing a disease or disorder associated with thepresence or accumulation of advanced glycation end products, the methodcomprising administering to a subject in need thereof a compositioncomprising as active ingredient a peptide of the present invention or asalt thereof. According to certain embodiments, the method comprisesadministering mixtures of peptides of the invention.

In some embodiments, the present invention provides the use of a peptideof the present invention or a salt thereof for the manufacture of amedicament for the treatment and/or prevention of a disease or disorderassociated with the presence or accumulation of advanced glycation endproducts.

In some embodiments, a pharmaceutical composition is provided,comprising at least one peptide of the present invention or at least onesalt thereof as an active ingredient, for use in the treatment and/orprevention of diseases or disorder associated with the presence oraccumulation of advanced glycation end products.

Examples of diseases and disorders associated with advanced glycationend products that may be treated using the peptides of the presentinvention include, but are not limited to, diabetes (type I and typeII). cataract, heart failure, hypertension and callus (such as feetcallus).

In some embodiments, the present invention provides the use of a peptideof the present invention or a salt thereof as an anti-inflammatoryagent.

In some exemplary embodiments, the present invention provides the use ofthe peptide Cys-Lys-Met-Cys (SEQ ID NO: 1) as an anti-inflammatoryagent. In additional exemplary embodiments, the present inventionprovides the use of the peptide Cys-Met-Lys-Cys (SEQ ID NO: 2) as ananti-inflammatory agent. In yet additional exemplary embodiments, thepresent invention provides the use of the peptide Cys-β-Ala-His-Cys (SEQID NO: 3), as an anti-inflammatory agent.

In some embodiments, the present invention provides the use of a peptideof the present invention or a salt thereof as an anti-allergic agent.

In some exemplary embodiments, the present invention provides the use ofthe peptide Cys-Lys-Met-Cys (SEQ ID NO: 1) as an anti-allergic agent. Inadditional exemplary embodiments, the present invention provides the useof the peptide Cys-Met-Lys-Cys (SEQ ID NO: 2) as an anti-allergic agent.In yet additional exemplary embodiments, the present invention providesthe use of the peptide Cys-β-Ala-His-Cys (SEQ ID NO: 3), as ananti-allergic agent.

In some embodiments, the present invention provides the use of a peptideof the present invention or a salt thereof as an anti-oxidant.

In some exemplary embodiments, the present invention provides the use ofthe peptide Cys-Met-Lys-Cys (SEQ ID NO: 2) as an anti-oxidant. Inadditional exemplary embodiments, the present invention provides the useof the peptide Cys-β-Ala-His-Cys (SEQ ID NO: 3), as an anti-oxidant.

According to another aspect, the present invention provides a method fortreating and/or preventing a disease or disorder associated with thepresence or accumulation of metal ions, the method comprisingadministering to a subject in need thereof a pharmaceutical compositioncomprising as an active ingredient a peptide of the present invention ora salt thereof.

In some embodiments, the present invention provides the use of a peptideof the present invention or a salt thereof for the manufacture of amedicament for the treatment and/or prevention of a disease or disorderassociated with the presence or accumulation of metal ions.

In some embodiments of the present invention, a pharmaceuticalcomposition is provided, comprising at least one peptide of the presentinvention or at least one salt thereof as an active ingredient, for usein the treatment and/or prevention of a disease or disorder associatedwith the presence or accumulation of metal ions.

Examples of diseases and disorders associated with the presence oraccumulation of metal ions that may be treated using the peptides of thepresent invention include, but are not limited to, a liver disorder,Alzheimer's disease, heart attack (myocardial infarction), stroke and aprion disease.

The cosmetic industry is constantly looking for new and improvedcompounds for skin care, particularly for compounds having anti-agingeffects. The properties of the peptides of the present invention may beutilized in cosmetic applications, for skin care and protection. Forexample, the peptides of the present invention may be utilized asanti-aging agents.

Thus, according to yet another aspect, the present invention provides amethod for slowing the aging process of the human skin, reducing thesigns of aging of the human skin or both, the method comprising applyingto the skin a cosmetic composition comprising the peptide of the presentinvention or a salt thereof.

In some embodiments, slowing the aging process of the human skin andreducing the signs of aging of the human skin encompass improvement ofthe skin tone, reduction of wrinkles, removal of lines, promotion ofskin firmness and reduction of skin sensitivity and irritability.

In some embodiments, a cosmetic composition is provided, comprising apeptide of the present invention or a salt thereof as active ingredient,for use in slowing the aging process of the human skin, reducing thesigns of aging of the human skin or both.

In additional embodiments, a cosmetic composition is provided,consisting of a peptide of the present invention or a salt thereof asactive ingredient, for use in slowing the aging process of the humanskin, reducing the signs of aging of the human skin or both.

The peptides of the present invention may be utilized in the preventionand/or treatment of damages caused by ionizing radiation.

In some exemplary embodiments, the present invention provides the use ofthe peptide Cys-Lys-Met-Cys (SEQ ID NO: 1) in the prevention ortreatment of damages caused by ionizing radiation. In addition exemplaryembodiments, the present invention provides the use of the peptideCys-Met-Lys-Cys (SEQ ID NO: 2) in the prevention or treatment of damagescaused by ionizing radiation. In yet additional exemplary embodiments,the present invention provides the use of the peptide Cys-β-Ala-His-Cys(SEQ ID NO: 3) in the prevention or treatment of damages caused byionizing radiation.

Thus, according to another aspect, the present invention provides amethod for the prevention and/or treatment of disorders and harmfuleffects caused by ionizing radiation, the method comprisingadministering to a subject in need thereof a composition, comprising apeptide of the present invention or a salt thereof. According to oneembodiment, the method comprises administering mixtures of peptides ofthe present invention.

“Ionizing radiations” as used herein include but are not limited tox-rays radiation, alpha radiation, beta radiation, gamma radiation,ultraviolet (UV) radiation, solar radiation, thermal radiation, nuclearradiation, energetic electron radiation, neutron radiation, positronradiation, cosmic radiation, electromagnetic radiation or a combinationthereof.

The compositions disclosed herein are also useful in preventing DNAdamage which may occur following ionizing radiation exposure. Withoutwishing to be bound by any theory or mechanism, it has been postulatedthat UV-B radiation act on DNA through direct excitation process of thenucleobases and further reactions proceed in an oxygen-independentmanner. This leads mostly to the formation of dimeric photoproducts atbipyrimidine sites in a strong sequence dependence manner. Thephotoproducts mostly involve cis-syn cyclobutadipyrimidines (CPDs) andpyrimidine (6-4) pyrimidone photoproducts (6-4PPs). Most of the damagingeffects of UV-A radiation on cellular DNA involve photosensitizationreactions.

“Ultraviolet (UV) radiation” is defined herein as a radiation between 10and 400 nanometers (nm) in wavelength. It is further characterized intoultraviolet A (315-400 nm), B (280-315 nm) and C (100-280 nm). The majorsource of UV radiation is sunlight, although exposure to artificialsources in the workplace, home and tanning salons are also a risk.Ultraviolet C (UV-C) coming from the sun is usually not harmful since itis screened out by the atmosphere's ozone layer. Although UV-B is animperative component in the synthesis of vitamin D, it is alsoresponsible for erythema (sunburn) skin cancers and immunosuppression.Ultraviolet A (UV-A) is responsible for skin aging and has additionallybeen implicated in the development of skin cancers in animals and inimmunosuppression in humans. The sun is the main source of UV-A, howeveruse of UV-A lamps for artificial tanning has also been shown to causeadverse side effects. The principal causes of morbidity and mortalityattributed to UV radiation are chronic diseases of the skin and the eyehowever other side effects such as erythema (sunburn), photoaging,photodermatoses, and the formation of nevus, amongst others, are alsoknown (Gllagher and Lee, Prog Biophys Mol. Biol. 2006, 92(1)).

The compositions disclosed herein are also useful in treating sideeffects and disorders associated with UV radiation exposure includingcutaneous conditions and ocular diseases linked to UV. Cutaneousconditions include but are not limited to malignant melanoma, basal andsquamous cell carcinoma (SCC) of the skin, cancers of the lip, cancersof the pinna (auricle) and non-melanocytic skin cancer (NMSC). Oculardiseases linked to UV radiation exposure include but are not limited touveal melanoma, cataract, macular degeneration, pterygium andphotokeratitis.

According to other embodiments the composition of the present inventionmay be incorporated into topical sun protecting formulations orsun-protecting compounds, namely any compound capable of blocking orreducing UV-A and/or UV-B radiation exposure to the skin, in order toenhance the effect of the sunblock and/or to reduce or prevent the skindamage. Examples of sun-protecting compounds include sunscreens(conventionally, products with a sun-protecting factor (SPF) of 2 orhigher), sunblocks (conventionally, products that physically blockradiation exposure and/or have an SPF of 12 or higher), and combinationsthereof.

The compositions disclosed herein are also useful in treating ionizingradiation damages which may occur following exposure to radiation inmedical procedures performed using medical imaging and medical scanningtechnologies, directed towards diagnosis or treatment of a disease or adisorder, which may be performed once or multiple times in the samepatient. Medical imaging and scanning technologies include but are notlimited to angiography, magnetic resonance imaging (MRI), magneticresonance angiography (MRA), functional magnetic resonance imaging(fMRI), magnetic resonance spectroscopy (MRS), computed tomography (CT),positron emission tomography (PET), positron emissiontomography-computed tomography (PET-CT), positron emissiontomography-fluorodeoxyglucose (PET-FDG), X-ray, ultrasound, dexa scan(bone D), echocardiography, fluoroscopy, mammography (and R2 imagechecker), radiolabel scans and nuclear medicine among others.

Medical procedures using imaging and scanning technologies and methodsinclude but are not limited to arthrogram, barium enema, barium swallow,modified barium swallow, hysterosalpinogogram, intra-venous pyelogram(IVP), uterine fibroid embolization (UFE), upper gastro-intestinalseries (UGI), voiding cystourethrogram (VCUG), aortography, cerebralangiography, coronary angiography, lymphangiography, pulmonaryangiography, ventriculography, chest photofluorography,echocardiography, electrical impedance tomography, fluoroscopy, diffuseoptical imaging, diffusion-weighted imaging, diffusion tensor imaging,positron emission tomography, scintillography, single photon emissioncomputed tomography (SPECT), gynecologic ultrasonography, obstetricultrasonography, contrast-enhanced ultrasound, intravascular ultrasound,thermography, virtual colonoscopy and general diagnostic X-ray examssuch as abdomen, chest, bones, and spine.

The compositions disclosed herein are also useful in treating and/orpreventing side effects and disorders associated with radiation therapy.Common side effects of radiation therapy include but are not limited tobone marrow damage, hair loss, fatigue, skin irritation, soreness in themouth, throat and esophagus, swelling, infertility and damage toepithelial surfaces such as skin, oral mucosa, pharyngeal, intestine andbowl mucosa, and ureter, all depending on the area of the body which hasbeen treated. Further side effects which can occur months to years aftertreatment with radiation therapy include but are not limited tofibrosis, epilation (hair loss), lymphedema, heart disease, cognitivedecline, radiation proctosis and most common, cancer.

The compositions disclosed herein are also useful in treating and/orpreventing side effects and disorders which may occur followingdiagnosis and/or treatment with nuclear medicine. Nuclear medicine usesradioactive pharmaceuticals that can localize and/or concentrate inspecific organs (or tissues) or cellular receptors. Nuclear medicineradioactive pharmaceuticals include but are not limited to calcium-47,carbon-11, carbon-14, choromium-51, cobalt-57, cobalt-58, erbium-169,fluorine-18, gallium-67, gallium-68, hydrogen-3, idium-111, iodine-123,iodine-125, iodine-131, iron-159, krypton-81m, nitrogen-13, oxygen-15,phosphorus-32, samarium-153, selenium-75, sodium-22, sodium-24,strontium-89, thallium-201, xenon-133, yttrium-90 and technetium-99m.

Workers and people living in the vicinity of a nuclear plant, medicalstaff of clinics and laboratories using specific technologies andmaterials, astronauts, pilots and aircrafts crews are constantly exposedto varying doses of ionizing radiation. Hence, the compositionsdisclosed herein are also useful in treating and/or preventing sideeffects and disorders which may occur following chronic or one timeexposure to nuclear radiation or other ionizing radiations. Furthermore,the compositions of the present invention are useful in treating and/orpreventing side effects and disorders which may occur following exposureto nuclear radiation of nuclear and radiation accidents or the use ofnuclear weapon.

The compositions disclosed herein are also useful in treating and/orpreventing adverse effects and disorders associated with the use ofcontrast agents (also known as medical contrast medium) in diagnosticand therapeutic medical procedures such as many kinds of imaging andscanning. An exemplary disorder associated with the use of iodinatedcontrast agents includes contrast-induced acute kidney injury (CI-AKI)(also known as contrast-induced nephropathy).

Diagnostic and therapeutic medical imaging procedures which use contrastagents include, but are not limited to, angiography, percutaneouscoronary intervention, venography, voiding cystourethrography (VCUG),hysterosalpinogram (HSG), intravenous urography (IVU), barium enema,double contrast barium enema (DCBE), barium swallow, barium meal, doublecontrast barium meal, barium follow through, virtual colonoscopy,contrast-enhanced CT, contrast-enhanced MRI, dynamic contrast-enhancedMRI and contrast-enhanced ultrasound (CEUS).

Contrast agents include, but are not limited to, radiocontrast agents,iodine, iodinated contrast agents, diatrizoate, metrizoate, ioxaglate,iopamidol, iohexyl, ioxilan, iopromide, iodixanol, barium, bariumsulfate, thorotrast, gadodiamide, gadobenic acid, gadopentetic acid,gadoteridol, gadofosveset, gadoversetamide, gadoxetic acid, gadobutrol,gadocoletic acid, gadodenterate, gadomelitol, gadopenamide, gadotericacid, cliavist, combidex, endorem (feridex), resovist, sinerem,perflubron, optison, levovist, microbubble contrast agents,gadolinium-containing contrast agents, iron-oxide contrast agents,Mn-DPDP and natural products such as blueberry and green tea.

According to other embodiments the composition is administered within 24hours before the exposure to the ionizing radiation or at about the timeof exposure to the ionizing radiation and/or within 24 hours after theexposure to the ionizing radiation. The pharmaceutical composition maybe administered in one or more doses within 24 hours before theradiation exposure or in one or more doses at about the time of theradiation exposure and/or in one or more doses within 24 hours after theradiation exposure. According to alternative embodiments the compositionis administered in one or more doses within 12 hours, within six hoursor within three hours or less before and/or after the exposure to theionizing radiation.

According to other embodiments the composition of the present inventionis administered one or more times before the radiation exposure and oneor more times after the radiation exposure. According to otherembodiments the composition may be administered one or more times within24 hours prior to the radiation exposure for example at about 1, 6, 12,18 or 24 hours prior to the radiation exposure and further administeredone or more times within 24 hours after the radiation exposure, forexample at about 1, 6, 12, 18 or 24 hours after the radiation exposure.

The amount of the pharmaceutical or cosmetic composition of the presentinvention to be administered for the above indications, theadministration regimes as well as their mode of application will dependboth on characteristics of the treated individual (age, size, gender,etc.) as well as on parameters associated with the phenomena to betreated.

The following examples are presented in order to more fully illustratecertain embodiments of the invention. They should in no way, however, beconstrued as limiting the broad scope of the invention. One skilled inthe art can readily devise many variations and modifications of theprinciples disclosed herein without departing from the scope of theinvention.

EXAMPLES Example 1 Peptide Synthesis

The following new five peptides were synthesized by Solid Phase PeptideSynthesis (SPSS) using Fmoc strategy (all five peptides were >98% pure):

(SEQ ID NO: 4) N-acetyl-Cys-Lys-Met-Cys-NH₂, designated herein as DY-65;(SEQ ID NO: 6) N-acetyl-Cys-βAla-His-Cys-NH₂,designated herein as DY-66; (SEQ ID NO: 13) N-acetyl-Cys-γGlu-Cys-NH₂,designated herein as DY-67; (SEQ ID NO: 5) N-acetyl-Cys-Met-Lys-Cys-NH₂,designated herein as DY-70;and

N-acetyl-Cys-γGlu-Cys-Cys-NH₂, designated herein as DY-71 (SEQ ID NO:12).

The peptides were prepared by SPSS in which there were repeated cyclesof coupling-deprotection. The first stage of the technique consisted ofpeptide chain assembly with protected amino acid derivatives on apolymeric support. The second stage of the technique was the cleavage ofthe peptide from the resin support with the concurrent cleavage of allside chain protecting groups to give the crude free peptide.

The free N-terminal amine of a solid-phase attached peptide was firstcoupled to a single N-protected amino acid unit. This unit was thendeprotected, revealing a new N-terminal amine to which a further aminoacid was attached. After cleavage from the resin, peptides were thenpurified by reverse phase HPLC using columns.

Fmoc Deprotection:

0.08 mmol of Fmoc-X-Wang resin was loaded into a fritted column equippedwith a plastic cap. The resin was washed twice with 3 mL portions ofdimethylformamide (DMF) for 1 minute each. Next, 3 ml of 20% piperidinein DMF was added and deprotection allowed to continue for 15 minutes.During this time, the column was gently swirled in order to assure acomplete mixing. After the reaction was complete (in about 15 minutes),the reaction column was drained and the resin washed 4 times with 3 mLof DMF.

Amide Bond Coupling:

In a small vial, 3 equivalents of the Fmoc amino acid was preactivatedby combining it with equal equivalents ofO-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate(HBTU), 6 equivalents of DIPEA (N,N′-diisopropylethylamine), and 3 mL ofDMF. This solution was fully dissolved and then allowed to react for anadditional 3-5 minutes. Then this coupling solution was added to theresin. The cap was placed on the reaction column and the resin slurryagitated every 2-3 minutes over a period of 20 minutes.

Cleavage:

In order to obtain the peptide in the free acid form, the ester linkagewas cleaved using trifluoroacetic acid (TFA). The resin was treated with2-3 mL of a solution of TFA and water in a ratio of 95:5. The resin wasthen agitated over a period of 25 minutes. The column was subsequentlydrained and the filtrated collected into a glass collection vessel. Thematerial was then dried in diethyl ether and analyzed.

In addition, the following peptides, known as CB3 and CB4, were alsosynthesized by SPSS using Fmoc strategy:

(SEQ ID NO: 14) CB3: N-acetyl-Cys-Pro-Cys-NH₂ (SEQ ID NO: 15)CB4: N-acetyl-Cys-Gly-Pro-Cys-NH₂

Example 2 Effect of the Peptides on Carrageenan-Induced Hind-PawSwelling

The above new five peptides were examined for anti-inflammatoryactivity, and their activity was compared to that of peptide CB3. MaleICR mice received an i.p. administration of each of the tested peptide(150 mg/kg) 30 min prior to the injection of carrageenan into thesub-plantar area of both of the animal's hind paws. Carrageenan wasdissolved in water at concentration of 3 mg/ml and injected at a volumeof 50 μl. Hind paw thickness was measured by Mitutoyo® micrometer beforecarrageenan injection and afterward at intervals of 1 hour between eachmeasurement. Swelling was calculated as the difference between thethickness of the hind paw measured at each time interval and its size attime 0 (before carrageenan injection). n=6 foot pads (3 animals) foreach group.

As can be seen in FIG. 1, the most active peptide was DY-65, whichshowed swelling reduction of 40%, 27% and 34% after 1, 2, and 3 hours,respectively, compared to control (saline only). Reduced swellingcompared to control was also observed for DY-66, DY-67, and to a lesserextent for DY-70 and DY-71. CB3 did not show a significant reductioncompared to control.

Example 3 Effect of the Peptides on Lipid Peroxidation in the Lung in aMurine Model of Zymosan-Induced Inflammation

Zymosan-Induced Shock.

Male BALB/c mice were randomly allocated into the following experimentalgroups (8 mice for each group):

(1) Zymosan+vehicle group (“ZYM”). Mice were treated intraperitoneallywith zymosan 500 mg/kg.

(2) Zymosan+DY-66 group (“ZYM+DY-66”). Identical to the Zymosan+vehiclegroup but peptide DY-66 was administered.

(3) Zymosan+DY-65 group (“ZYM+DY-65”). Identical to the Zymosan+vehiclegroup but peptide DY-65 was administered.

(4) Zymosan+DY-70 group (“ZYM+DY-70”). Identical to the Zymosan+vehiclegroup but peptide DY-70 was administered.

(5) Zymosan+DY-71 group (“ZYM+DY-71”). Identical to the Zymosan+vehiclegroup but peptide DY-71 was administered.

(6) Zymosan+DY-67 group (“ZYM+DY-67”). Identical to the Zymosan+vehiclegroup but peptide DY-67 was administered.

(7) Sham group (“Sham”). Identical to the Zymosan+vehicle group but avehicle was administered instead of zymosan.

The peptides were administered by an intraperitoneal (i.p.) route ofadministration as follows:

-   -   The first dose (50 mg per kg) was given 3 days before the        zymosan injection.    -   The second dose (50 mg per kg) was given 2 days before the        zymosan injection.    -   The third dose (50 mg per kg) was given 1 day before the zymosan        injection.    -   The fourth dose (120 mg per kg) was given 30 minutes before the        zymosan injection.    -   The fifth dose (120 mg per kg) was given 5 hours after the        zymosan injection.

The peptides were formulated in D5W (5% dextrose in water, “D5W”). Thevehicle control was D5W. At 24 hours after administration of zymosan,animals were euthanized and assessed for inflammation by an MDA(malondialdehyde) analysis of lung tissue. All the materials and drugs(apart from the peptides) were obtained from Sigma, Inc.

Determination of MDA Activity.

Malondialdehyde (MDA) formation was used to quantify lipid peroxidation,measured as thiobarbituric acid-reactive material. Tissues werehomogenized (100 mg/mL) in 1.15% KCl buffer and homogenates (200 mL)were then added to a reaction mixture consisting of 0.75 mL 0.8%thiobarbituric acid, 100 mL 8.1% (volume per volume) sodium dodecylsulfate, 0.75 mL 20% (v/v) acetic acid (pH 3.5) and 300 mL dH₂O, andheated at 95° C. for 60 min. After cooling to room temperature, sampleswere cleared by centrifugation at 10,000 g for 10 min and absorbance wasmeasured at 532 nM using 1,1,3,3-tetramethoxypropane as an externalstandard. The level of lipid peroxides was expressed as nM MDA/mgprotein.

Data Analysis.

All values in FIG. 2 and the text below were expressed as mean±standarddeviation (SD) and standard error (SE) of n observations. For the invivo studies n represents the number of animals studied. The resultswere analyzed by one-way ANOVA followed by a Bonferroni's post-hoc testfor multiple comparisons. A p-value of less than 0.05 was consideredsignificant.

Effect of the Peptides on MDA Activity.

As can be seen in FIG. 2, zymosan administration induced a 50% increasein MDA activity in the lung tissue at 24 hours after zymosanadministration. Treatment with peptide DY-70 inhibited the elevation inMDA concentration in the lung tissue (p<0.05).

Example 4 Effect of the Peptides on Nuclear Translocation of NF-kappaBand Cytoplasmic Stability of I-Kappa-Balpha in the Lung in a MurineModel of Zymosan-Induced Inflammation

The following peptides were examined in this Example: DY-65, DY-66 andDY-70.

Zymosan-Induced Shock.

Male BALB/c mice were randomly allocated into the following experimentalgroups:

(1) Zymosan+vehicle group (“ZYM”). Mice were treated intraperitoneallywith zymosan 500 mg/kg.

(2) Zymosan+DY-65 group (“ZYM+DY-65”). Identical to the Zymosan+vehiclegroup but peptide DY-65 was administered.

(3) Zymosan+DY-66 group (“ZYM+DY-66”). Identical to the Zymosan+vehiclegroup but peptide DY-66 was administered.

(4) Zymosan+DY-70 group (“ZYM+DY-70”). Identical to the Zymosan+vehiclegroup but peptide DY-70 was administered.

(5) Sham group (“SHAM”). Identical to the Zymosan+vehicle group but avehicle was administered instead of zymosan.

The peptides were administered by an intraperitoneal (i.p.) route ofadministration as described in Example 3.

The peptides were formulated in D5W as described in Example 3. Thevehicle control was D5W.

At 24 hours after administration of zymosan, animals were euthanized andassessed for inflammation by an NF-κB analysis of lung tissue.

Determination of Nuclear Translocation of NF-κB.

Tissues were homogenized and nuclear extracts prepared viacentrifugation. ELISA for p50 was performed, using a series of standardsin order to define absolute concentrations.

Determination of Cytoplasmic Stability of I-κBalpha.

Tissues were homogenized and nuclear extracts prepared viacentrifugation. ELISA for IkappaBalpha was performed, using a series ofstandards in order to define absolute concentrations.

Data Analysis.

All values in FIG. 3 and the text below were expressed as mean±standarddeviation (SD) and standard error (SE) of n observations. For the invivo studies n represents the number of animals studied. The resultswere analyzed by one-way ANOVA followed by a Bonferroni's post-hoc testfor multiple comparisons. A p-value of less than 0.05 was consideredsignificant.

Effect of the Peptides on Nuclear Translocation of NF-κB.

As can be seen in FIG. 3A-F, Zymosan (“ZYM”) administration induced a10-fold increase in p65 nuclear translocation in the lung tissue at 24hours after zymosan administration. Treatment with peptide DY-65 orDY-70 inhibited the increase in nuclear p65 translocation by >80%(p<0.05). Treatment with peptide DY-66 inhibited the increase in nuclearp65 translocation by ca. 80% (p<0.05).

Effect of Peptides on Cytoplasmic Stability of I-kappaBalpha.

Zymosan administration induced a 4-fold decrease in cytoplasmicIkappaBalpha concentration in the lung tissue at 24 hours after zymosanadministration. Treatment with peptides DY-65, DY-66 or DY-70 inhibitedthe decrease in cytoplasmic IkappaBalpha concentrations by >50-80%(p<0.05 for all three peptides), with efficacy of DY-70>DY-65>DY-66.

Example 5 Reversal of Oxidative Stress-Induced Phosphorylation of p38and JNK by the New Peptides

All five peptides were examined in this Example according to thefollowing procedure:

Cell Culture and Treatment:

Human neuroblastoma SH-SY5Y cells were maintained in Dulbecco's modifiedEagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovineserum (Invitrogen), 100 μg/ml streptomycin and 100 U/ml penicillin G(Gibco). Cells were plated at a density of 6.25×10⁴/cm² and incubatedfor 24 hours, after which they were exposed to different treatments.

Cell Viability:

SH-SY5Y cells were plated on 96-well plates and treated with Auranofin(AuF) (5 μM) for 30 min. Then the cells were washed with PBS and treatedwith either one of the peptides at the indicated concentrations.Twenty-four hours later, the cells were fixed with glutaraldehyde infinal concentration of 0.5% for 10 min. Cells were washed 3 times withDDW dried over night, and washed once with borate buffer (0.1M, pH 8.5).The fixed cells were stained with 200 μl of 1% methylene blue dissolvedin borate buffer for 1 hour. After extensive washing and drying, thecolor was extracted with 200 μl of 0.1 M HCl for 1 h at 37° C. andabsorbance was read in spectrophotometer at 630 nm.

Western Blot Analysis and Antibodies:

Twenty to thirty micrograms of protein samples were loaded on 10-12%SDS-PAGE gels. The proteins were then transferred electrophoretically tonitrocellulose (Whatman, Germany). The blots were blocked by incubationfor 1 h at room temperature (RT) in TBS-T (25 mM Tris-HCl pH 7.4, 0.9%NaCl and 0.02% Tween-20) with 4% Difco skim milk (BD, USA), andincubated over-night at 4° C. with the primary antibody: pERK1/2 (Thr202/Tyr204), mouse mAb, (1:6,000); p-SAPK/JNK (Thr183/Tyr185), rabbitmAb; SAPK/JNK, mouse mAb; p-p38MAP Kinase (Thr180/Tyr182), rabbit mAb;cleaved caspase 3, rabbit mAb. All antibodies were from Cell SignalingTech. USA, used at 1:1000. Purified b Catenin, mouse mAb, (1:10,000; BDTransduction Laboratories, USA) diluted in 5% BSA, 0.04% Azide in TBS-T.Proteins were detected with Anti-Mouse or Anti-Rabbit IgG-HRP linkedantibody (1:10,000; Cell Signaling, Tech. USA).

The results are summarized in Table 1 hereinbelow. As can be seen fromthe table, all five peptides were highly effective in reducingphosphorylation of p38 MAP Kinase and JNK (c-Jun N-terminal kinases) atconcentrations in the range of 20-90 μM. DY-70 and DY-65 were the mostpotent.

TABLE 1 Inhibition of MAPK phosphorylation by the pepides JNKphosphorylation p38 phosphorylation Compound (IC₅₀) μM (IC₅₀) μM DY-6532.3 36.7 DY-66 86.9 67.0 DY-67 67.3 36.5 DY-70 22.1 22.2 DY-71 82.346.9

Example 6 Effect of the Peptides on Ovalbumin-Induced Hypersensitivityin Mice—Analysis of Blood and Peritoneal Exudates Samples

The following peptides were examined in this Example: DY-65, DY-66 andDY-70.

Hypersensitivity.

Male BALB/c mice (6-8 weeks old) were housed under specificpathogen-free conditions and maintained on a 12-hour light/dark cycle,with food and water ad libitum. All materials and drugs were obtainedfrom Sigma, Inc.

Mice in Groups A, B, C, and E (n=10 per group) were sensitized withintraperitoneal (i.p.) 200 μl of sterile PBS containing 100 μg ovalbumin(OVA) and 1.6 mg alum on days 0 and 7, and challenged withintraperitoneal (i.p.) 200 μl of sterile PBS containing 10 μg ovalbumin(OVA) on day 14 in order to induce an experimental model of allergicperitonitis. Mice in Group F (n=10) were injected with intraperitoneal(i.p) 200 μl sterile PBS (“sham” group, no OVA and no Alum).

Mice in Groups A, B, and C, were treated with 150 μl of sterile PBScontaining peptides DY-66, DY-65 and DY-70, respectively, at a dose of125 mg/kg on days 13, 14, and 15 via an i.p. route of administration.

Mice in Groups E and F were treated with 150 μl of sterile PBS on days13, 14, and 15 via an i.p. route of administration. On day 14 the testarticles (or PBS vehicle) were injected 30 minutes before injecting theOVA.

Blood taken on day 16 was tested for 1) serum anti-OVA IgE, 2) whiteblood cell count and differential (including absolute and percentage ofeosinophils count). Serum was frozen at −80° C. for future optionalblood tests.

Data Analysis.

All values in FIG. 4 and the text below were expressed as mean±standarddeviation (SD) and standard error (SE) of n observations. For the invivo studies n represents the number of animals studied. The resultswere analyzed by one-way ANOVA followed by a Bonferroni's post-hoc testfor multiple comparisons. A p-value of less than 0.05 was consideredsignificant.

Effect of Peptides on Peritoneal Cellularity.

As can be seen in FIG. 4A, OVA immunization (“Ova+vehicle”) induced anearly 7-fold increase in the cellularity of the peritoneal exudates.All three peptides significantly reduced the elevation in thiscellularity, as follows: DY-65>DY-70>DY-66. The effect on macrophage andeosinophil cellularity in the peritoneal exudate followed the same rankordering (FIG. 4B).

Effect of Peptides on Serum Anti-OVA IgE Levels.

As can be seen in FIG. 4C, OVA immunization induced a nearly 2.5-foldincrease in the titer of anti-OVA serum IgE. All three peptidessignificantly reduced the elevation in this titer, as follows:DY-65>DY-70>DY-66, with the greatest reduction ca. 60% of theOVA-induced elevation.

Example 7 Effect of the Peptides on Ovalbumin-Induced Hypersensitivityin Mice—Analysis of Peritoneal Exudate Samples

In this example, peptide DY-66 was examined and compared to the knownCB3 peptide.

Hypersensitivity was induced as described in Example 5. The mice weretreated according to the above protocol with PBS alone (“PBS”, no OVAand no Alum), OVA (“OVA”), OVA+CB3 104 mg/kg, OVA+DY-66 83 mg/kg andOVA+DY-66 125 mg/kg. In all groups, 9 mice were used except forOVA+DY-66 83 mg/kg, where 10 mice were used.

At day 16 the animals were sacrificed, the peritoneum was washed with4-5 ml of sterile PBS, centrifuged and the cells were re-suspended insterile PBS containing FCS 2%.

Total White Blood Cells (WBC) Counts.

Total WBC were collected by peritoneal washing (where needed, red bloodcells, RBC, were lysed) and analyzed by FACS machine to count total cellnumbers during 10 seconds flow. Averages and STDEV are shown in FIG. 5A.All results+medians are shown in FIG. 5B. Medians are marked byhorizontal lines.

Percentage of Total Granulocytes.

Percentage of granulocytes at the peritoneal lavages were measured bythe use of FACS machine analysis of all cells during 10 second flow,followed by a careful analysis of the FACS dot plots. Averages+STDEV areshown in FIG. 5C, and all results+medians are shown in FIG. 5D. Exceptfor the difference between PBS and OVA that was found significant, alsothe difference between medians of OVA and OVA+DY-66 125 mg/kg was foundto be highly significant, according to Mann-Whitney test+the stringentcorrection of Bonfferoni (p<=0.0125 (0.05/4)).

Eosinophil (Eos.) Percentage

About 100,000 cells were taken from each sample and “blotted” on a slideby the use of a cytospin machine. Slides were stained with“Wright-Giemsa” stain solution for identification of eosinophils, mastcells, other WBC and RBC. Cells were counted and % of eos wascalculated. Averages and STDEV are shown in FIG. 5E. All results+mediansare shown in FIG. 5F. Medians are marked by horizontal lines.

Absolute Numbers of Eos/ml.

Total eos. numbers were calculated by multiplying total WBC counts byeos. % (divided by 100). Averages and STDEV are shown in FIG. 5G. Allresults and medians are shown in FIG. 5H. Medians are marked byhorizontal lines.

Example 8 Effect of the Peptides on Hydrogen Peroxide-Induced HaCatKeratinocyte Cytotoxicity

All five peptides were examined in this Example, and their activity wascompared to the known CB3 peptide. HaCaT cells were seeded in 96-wellplate at a concentration of 10×10⁴ cells/ml for 24 hours. Then cellswere incubated with the peptides at the indicated concentrations for 2.5hours followed by washing with PBS and incubation with 90 μM hydrogenperoxide (without peptide) for 30 minutes. Cells were washed with PBSand incubated with culture medium containing peptide for 72 hours. Cellviability was tested by MTT. The effect of the peptides on cellviability was performed similarly without hydrogen peroxide exposure.

The results are summarized in FIG. 6. “Control HaCaT”—cells that wereneither treated by peptide nor by hydrogen peroxide; “Medium”—wellwithout cells that contained only culture medium; “HaCaT H₂O₂”—cellstreated with hydrogen peroxide.

The results have shown that peptide DY-70 fully protected the cells fromhydrogen peroxide at all tested concentrations (FIGS. 6A—no hydrogenperoxide; 6B—after hydrogen peroxide exposure). Peptide DY-67 causedincrease of about 45% in cell viability at concentrations of 1-10 μM(FIGS. 6C—no hydrogen peroxide; 6D—after hydrogen peroxide exposure).Peptides DY-65, DY-66, DY-71 and CB3 slightly elevated cell viability atselected concentrations after hydrogen peroxide exposure.

Example 9 Effect of DY-65 on Carnitine Levels in Mouse Tissues

Male ICR mice were injected i.p. with 250 mg/kg DY-65 for 5 consecutivedays. Half an hour after the last injection lung, heart, skeletal muscle(femural), brain, liver, kidney and blood were removed, homogenized (100mg tissue in 200 μl distilled water), centrifuged and filtrated through30K Microcon® filter. Carnitine analysis was carried out by a commercialkit (BioVision, catalogue number K642-100). n=4 for each group.

The results have shown a significant elevation (about 150% increase) inthe level of carnitine in some tissues. In other tissues, an elevationin the level of carnitine was observed (about 70% increase). The resultswere verified in pooled tissues, as well as in individual animals.

Example 10 Effect of the Peptides on UVB-Induced Cell Cytotoxicity inHaCaT Cells

The following peptides were examined in this Example and their activitywas compared to the known CB3 peptide: DY-65, DY-66 and DY-70.

HaCaT cells were seeded in 96-wells plate (10×10⁴/ml) and incubated in37° C., 6% CO₂ for 24 hours. Cells were incubated with the peptides atthe indicated concentrations for 2.5 hours and washed (PBS). Then 100 μlPBS were added to each well followed by UVB irradiation (0.05 J/cm²).Cells were washed with PBS and incubated with culture medium (DMEM, 100μl) containing the peptides. MTT assay was carried out after 72 hours.

The results are summarized in FIGS. 7A-C. “Control”—cells that wereneither treated by peptide nor by UVB irradiation; “Medium”—well withoutcells that contained only culture medium; “HaCaT UVB”—cells treated withUVB irradiation.

The results have shown that peptides DY-65 (FIG. 7A), DY-66 (FIG. 7B)and DY-70 (FIG. 7C) showed protective effect at low concentrations.

Example 11 Effect of DY-66 on ROS Levels in C57Bl/6 Mice afterLipopolysaccharide (LPS) Injection

C57BL/6 female mice (7-8 weeks old) were injected i.p. with the peptideor PBS on days −3, −2, −1 at a dose of 50 mg/kg or with PBS alone (6mice in each group). On day zero all the mice were injected i.p. with200 μg/mouse of LPS and 45 min. later they were injected i.p. with 150mg/kg of the peptide. Another group of 3 mice served as normal untreatedmice. Six hours after LPS injection mice were bled into heparinizedtubes (approx. 300 μl of blood from each mouse) and the level of ROS wasmeasured in the plasma.

The results have shown a significant reduction in ROS levels inplatelets and RBCs of mice treated with peptide DY-66 compared toPBS-treated mice.

Platelets: ROS level in DY-66-treated mice was approximately 55% of thelevel measured in PBS-treated mice (p<0.005).

RBCs: ROS level in DY-66-treated mice was approximately 75% of the levelmeasured in PBS-treated mice (p<0.05).

Example 12 Effect of the Peptides on Liver Inflammation in C57BL/6 Mice

First, all five peptides were examined in a mouse model of liverinflammation induced by injection (i.v.) of Concanavalin A (Con-A).

Six groups of C57BL/6 female mice (7-8 weeks old) containing 6 mice ineach group were injected i.p. with different peptide on days −3, −2, −1at a dose of 50 mg/kg or with PBS alone. On day zero all the mice wereinjected i.v. with 250 μg/mouse of Con A and 45 min. later they wereinjected i.p. with 150 mg/kg of each peptide or PBS.

Eight hours after Con A injection mice were bled into heparinized tubes(approx. 150 ul of blood from each mouse) and the levels of alanineaminotransferase (ALT) was measured in the plasma.

One day later the mice were sacrificed and their livers were placed in4% formalin and sent for pathological examination.

Administration of Con A induced a dramatic increase in ALT levels.Peptide DY-66 reduced ALT average by 69% and ALT median was reduced by75% compared to PBS-treated mice. In addition, treatment with DY-66resulted in significant (p=0.011) decrease in necrotic area according tohisto-pathological report.

Although the mice were injected with sub-lethal dose of Con A (250 μg/20g), 24 hours post Con A injection 3 mice of the PBS treated group and 1mouse of the DY-65 treated mice were found dead. There were nomortalities in the other groups. This suggests that most of the peptideshad some beneficial effect over Con A induced liver inflammation.

Next, peptide DY-66 was examined and compared to N-acetylcysteine (NAC),CB3 and CB4. The compounds were tested according to the proceduredescribed above. ConA dose was 180 μg/mouse.

FIG. 8 presents the change in ALT levels (%) for each of the testedcompounds compared to PBS treatment (i.e., the ratio between ALT levelsin samples obtained from mice treated with a tested compound and ALTlevels in samples obtained from PBS-treated mice)

Peptide DY-66 reduced ALT average by 71% and ALT median was reduced by89% compared to PBS-treated mice. Both CB3 and CB4 resulted in highlyincreased ALT level compared to PBS-treated mice. NAC did not induce asignificant change in ALT level compared to PBS-treated mice.

Example 13 Metal Chelation

All five peptides were examined for their ability to bind copper andferric ions. 0.83 mM of each peptide were mixed with 1.66 mM of eitherFeCL₃ or CuSO₄. The mixtures were incubated for 1 hour at roomtemperature and further kept at ° 4 C until analysis. Samples wereanalyzed by mass spectra (ESI).

Cupper binding was observed for DY-65, DY-66 (low degree) and DY-70 (lowdegree). FeCl binding was observed for DY-66 and DY-70 (low degree).

Example 14 Effect of Peptide on the Level of Advanced GlycationEnd-Products

Scope of work: establish the in vitro efficacy of a tested peptide (forexample, DY-66) in suppressing development of advanced glycation endproducts in endothelial cells incubated in a high glucose medium.

Bovine aortic endothelial cells are cultured for 7 days in MEM with 30mM glucose and 0.4% fetal bovine serum, with daily change of media.These conditions have been previously shown to induce a 261% increase inthe concentration of advanced glycation end products.

Prior to exposure to 30 mM glucose, cells are randomly allocated to thefollowing experimental groups:

1. Sham (5 mM glucose; no exposure to 30 mM glucose)

2. Vehicle control (30 mM glucose)

3. Peptide (40, 100, or 400 μM)+30 mM glucose

4. Carnosine (40, 100, or 400 μM)+30 mM glucose

5. N-acetylcysteine (NAC) (40, 100, or 400 μM)+30 mM glucose

Each condition is represented in triplicate. Given 10 conditions, and 3replicates per condition, a total of 30 wells are incubated.

After 7 days of incubation, cells are harvested and cytosol extractedfor dot-blot analysis of immunoreactive AGE using acommercially-available monoclonal antibody.

Blots are developed and densitometry performed. The IC50's of the testedpeptide and carnosine are determined and expressed as mean±standarderror.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed chemical structures andfunctions may take a variety of alternative forms without departing fromthe invention.

The invention claimed is:
 1. A peptide having the amino acid sequenceselected from the group consisting of Cys-Lys-Met-Cys (SEQ ID NO: 1),Cys-Met-Lys-Cys (SEQ ID NO: 2), and Cys-β-Ala-His-Cys (SEQ ID NO: 3),wherein the peptide is a tetra-peptide, further comprising at least onemodification selected from the group consisting of an amino-terminalmodification and a carboxy-terminal modification.
 2. The peptide ofclaim 1, wherein the amino-terminal modification is selected from thegroup consisting of an amino-terminal blocking group; and apermeability-enhancing moiety selected from the group consisting oflipids, fatty acids, steroids and bulky aromatic or aliphatic compounds.3. The peptide of claim 2, wherein the amino-terminal blocking group isselected from the group consisting of acetyl, alkyl and acyl.
 4. Thepeptide of claim 2, wherein the permeability-enhancing moiety is a fattyacid.
 5. The peptide of claim 4, wherein the fatty acid is selected fromthe group consisting of palmitic acid, phosphatidic acid, stearic acid,arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid and oleicacid.
 6. The peptide of claim 1, wherein the carboxy-terminalmodification is a carboxy-terminal blocking group.
 7. The peptide ofclaim 6, wherein the carboxy-terminal blocking group is selected fromthe group consisting of amide, ester and alcohol group.
 8. The peptideof claim 1, wherein the peptide is in the form of a salt.
 9. The peptideof claim 8, wherein the salt is selected from the group consisting oftrifluoroacetic acid (TFA), acetate and citrate.
 10. A compositioncomprising a peptide or a salt thereof according to claim
 1. 11. Thecomposition of claim 10, wherein the composition is formulated fortopical administration.
 12. A method for treating a disease or disorderassociated with oxidative stress, the method comprising administering toa subject in need thereof a pharmaceutical composition comprising asactive ingredient a peptide or a salt thereof according to claim 1,wherein the disease or disorder associated with oxidative stress isselected from the group consisting of Alzheimer's disease, Parkinson'sdisease, diabetes, rheumatoid arthritis, ischemia-reperfusion injury,contrast-induced nephropathy and vitiligo.
 13. A method for treating adisease or disorder associated with inflammation, the method comprisingadministering to a subject in need thereof a pharmaceutical compositioncomprising as active ingredient a peptide or a salt thereof according toclaim 1, wherein the disease or disorder associated with inflammation isselected from the group consisting of acute inflammation, rheumatoidarthritis and atherosclerosis.
 14. A method for treating a disease ordisorder associated with allergy, the method comprising administering toa subject in need thereof a pharmaceutical composition comprising asactive ingredient a peptide or a salt thereof according to claim 1,wherein the disease or disorder associated with allergy is selected fromthe group consisting of an allergic airway disease, asthma, allergicrhinitis, eczema, dermatitis, a gastrointestinal food allergy and anocular allergy.
 15. A method for treating a disease or disorderassociated with the presence or accumulation of advanced glycation endproducts, the method comprising administering to a subject in needthereof a pharmaceutical composition comprising as active ingredient apeptide or a slat thereof according to claim 1, wherein the disease ordisorder associated with the presence or accumulation of advancedglycation end products is selected from the group consisting ofdiabetes, cataract, hypertension and callused skin.
 16. A method forslowing the aging process of the human skin, reducing the signs of agingof the human skin or both, the method comprises applying to the skin acosmetic composition comprising as active ingredient a peptide or a saltthereof according to claim
 1. 17. A method for treating harmful effectscaused by ionizing radiation associated with radiation therapy selectedfrom the group consisting of bone marrow damage, fatigue, skinirritation, soreness in the mouth, throat and esophagus, swelling,damage to epithelial surfaces, fibrosis, lymphedema, heart disease, andradiation proctitis, the method comprising administering to a subject inneed thereof a composition comprising as active ingredient a peptide ora salt thereof according to claim
 1. 18. The composition of claim 10,wherein the composition is formulated for systemic administration.
 19. Amethod for treating a disorder caused by ionizing radiation, the methodcomprising administering to a subject in need thereof a compositioncomprising as active ingredient a peptide or a salt thereof according toclaim 1, wherein the disorder is a coetaneous condition selected fromthe group consisting of malignant melanoma, basal and squamous cellcarcinoma (SCC) of the skin, lip cancer, cancer of the auricle andnon-melanocytic skin cancer (NMSC), or an ocular disease selected fromthe group consisting of uveal melanoma, cataract, macular degeneration,pterygium and photokeratitis.
 20. A method for treating a disease ordisorder associated with the presence or accumulation of metal ions, themethod comprising administering to a subject in need thereof acomposition comprising as active ingredient a peptide or a salt thereofaccording to claim 1, wherein the disease or disorder is selected fromthe group consisting of iron overload and copper overload.